Synthesis and Anti-Melanoma Activity of Acryloyl Pyridinone Analogues

Abstract

A major challenge for clinical management of melanoma is the prevention and treatment of metastatic disease. Drug discovery efforts over the last 10 years have resulted in several drugs that improve the prognosis of metastatic melanoma; however, most patients develop early resistance to these treatments. We designed and synthesized, through a concise synthetic strategy, a series of hybrid olefin-pyridinone compounds that consist of structural motifs from tamoxifen and ilicicolin H. These compounds were tested against a human melanoma cell line and patient-derived melanoma cells that had metastasized to the brain. Three compounds 7b, 7c, and 7g demonstrated promising activity (IC50=0.4–4.3 μM). Cell cycle analysis demonstrated that 7b and 7c induce cell cycle arrest predominantly in the G1 phase. Both 7b and 7c significantly inhibited migration of A375 melanoma cells; greater effects were demonstrated by 7b. Molecular modelling analysis provides insight into a plausible mechanism of action.

Introduction

Melanoma, a malignancy that arises from cancerous melanocytes, is responsible for 75% of deaths related to skin cancer.^[1] While many cutaneous melanoma lesions are identified and removed early, a significant number of patients are diagnosed at advanced stages of the disease.^[2] Recently, therapeutics that modulate the host immune system, such as immune checkpoint inhibitors,^[3] have resulted in positive clinical outcomes for patients with advanced disease; however, approximately 50% of these patients either do not respond to the treatments or eventually develop secondary resistance.^[4] Because melanoma is not sensitive to radiotherapy and rapidly develops resistance when treated with molecularly targeted therapy, such as MEKi or MAPKi, there is a need for the development of novel therapeutics that can replace or complement existing therapies.^[5–6]

Tamoxifen, an estrogen receptor alpha (ER-α) antagonist, is often recommended to treat breast cancer.^[7] In a number of in vitro investigations, elevated levels of estrogen receptors were found on melanoma cells that originated from metastatic tumors.^[8] ER-α, in particular, promotes cell growth and cellular atypia.^[8] Thus, tamoxifen has been used as a single agent or, more frequently, in conjunction with other chemotherapy drugs to treat cases of metastatic malignant melanoma.^[9] Tamoxifen has been reported to prevent human melanoma and ovarian cancer cell lines from developing resistance to cisplatin,^[10] and it has been shown to increase sensitivity of resistant metastatic melanoma cell lines to curcumin.^[11]

Ilicicolin H demonstrates strong and broad antifungal effects via inhibiting the Qn site of the yeast cytochrome bc1 complex.^[12] It has also been recently reported to target Phosphoglycerate Kinase 1 (PGK1) to inhibit metabolic process of hepatocellular carcinoma cells (HCC) resulting in apoptosis of HCC cells.^[13] Jing Li et al. have utilized a cellular thermal shift assay (CETSA) and surface plasmon resonance (SPR) techniques to demonstrate that ilicicolin H binds to PGK1 in HepG2 cells promoting it’s apoptotic ability by inhibiting lactate production and glucose uptake.^[13] They have further established that ilicicolin H binds to the non-ATP sites in PGK1 and blocks the interactions between ATP and PGK1 critical for metabolic glycolysis pathway and energy supply. Furthermore, they have shown that, in contrast to control groups, ilicicolin H decreased HCC cell viability in a dose and time dependent manner by promoting apoptosis as evident from elevated cleaved caspase-3 and cleaved PARP proteins shown in western blots. Additionally, a recent report suggests that Vemurafenib’s effectiveness against melanoma cells is increased when PKG1 expression is silenced.^[14] Many malignancies, such as breast,^[15] hepatocellular carcinomas,^[13,16] and gastric,^[17] overexpress PGK1 to exhibit increased glycolysis and mitochondrial metabolism for energy supply. Altering cancer cell metabolism has emerged as an important strategy in preclinical and clinical studies making PGK1 an important target in cancer chemotherapy.

Prompted by these recent findings, we developed a set of tamoxifen and ilicicolin H derivatives as potential therapeutic agents for malignant melanoma. We designed the library as a set of hybrid compounds inspired by tamoxifen (compound 1), its closely related analog idoxifene (compound 2), and ilicicolin H (Figure 1).^[13,18–20] Close scrutiny of these molecules led us to identify 2 fragments that could potentially be assembled to generate hybrid scaffolds; the diaryl olefin moiety (in red, Figure 1) from tamoxifen (1) and idofen (2), and the pyridone motif (in blue, Figure 1) from ilicicolin-H (3). Next, the moieties were amalgamated to generate compound 7, which is a hybrid scaffold belonging to 2 classes of natural products/ pharmaceutical drugs (Figure 1). Our goal was to connect the polysubstituted olefin with the pyridone moiety via a carbonyl tether, and to incorporate nucleophilic functionalities (e.g., amine or amide) in the ring, which could make our molecules potentially more efficacious (Figure 1). Herein, we report the synthesis and anti-melanoma efficacy of polysubstituted olefinpyridinone type hybrid compounds.

Figure 1.

Design of hybrid molecules from polysubstituted olefins and pyridine-based natural products.

Results and Discussion

Taking cues from the retrosynthetic analysis, we began exploring appropriate conditions for the multicomponent reaction to synthesize olefin-pyridone hybrid 7 (Scheme 1). We reacted 4 (1 equivalent; synthesized from 2-cyanoacetamide and ethyl-2-ethoxyacetate) and 5, 2-tolylaldehyde (2 equivalents) with a variety of nucleophilic bases (3 equivalents) in various solvents.^[31–24] Liquid chromatography-mass spectrometry (LC–MS) was used to monitor the progress of the reactions. Nucleophilic bases included morpholine, piperidine, pyrrolidine and N-methylpiperazine (NMP). The solvents that were explored included tetrahydrofuran (THF), ethanol (EtOH), dioxane, dichloromethane, and dimethylformamide. With nucleophilic bases morpholine, pyrrolidine, and NMP at 40°C or under THF reflux afforded mostly the Knoevenagel condensation product 6 (11–41% conversion) (Table 1, entries 1–6). Interestingly, 9a and 8a were formed as the minor products (2–5% conversion) with nucleophilic bases morpholine and NMP (Table 1, entries 1–2 and 5–6). Finally reaction with nucleophilic base piperidine under THF reflux converted 4 to ~43% of 7a (Table 1, entry 8). Screening the reaction with piperidine in a variety of solvents (Table 1, entries 9–12) under reflux showed that EtOH was the most suitable solvent, generating 7a in 83% yield (~100% conversion) (Table 1, entry 12). Hence, for generating olefin-pyridone hybrid 7a, the optimized protocol includes reaction of 1 equivalent of furopyridinedione with 2 equivalents of tolylaldehyde and 3 equivalents of piperidine, under ethanol reflux.

Scheme 1.

Synthesis of compound 7 derivatives.

Table 1.

Reaction optimization for the domino multicomponent reaction.

Entry	Base-Nucle-ophile	Solvents	Temp (T °C)	Conversion
				B %	7–10
1	Morpholine	THF	40	11	2 (9a)
2	“	“	70	39	4 (9a)
3	Pyrrolidine	“	40	22	- (10a)
4	“	“	70	41	-
5	NMP	“	40	18	4 (8a)
6	“	“	70	31	5 (8a)
7	Piperidine	“	40	48	15 (7a)
8	“	“	70	18	43 (7a)
9	“	Dioxane	100	14	69 (7a)
10	“	Dichloromethane	45	49	21 (7a)
11	“	Dimethylformamide	120	8	55 (7a)
12	“	Ethanol	90	–	~100/ 83c (7a)

^[a]Conversion monitored with LC–MS; [c] Isolated yield of 7a.

After optimizing the reaction conditions, we next assessed the scope of the synthetic strategy with various aromatic (characterized by electron-withdrawing and electron-donating functionalities) and heteroaromatic aldehydes; results are shown in Figure 2. With all aldehydes, the protocol furnished products 7a–j in moderate to excellent yields (Figure 2). The reactions with electron-rich aromatic aldehydes resulted in synthesis of desired products 7a, 7c–h, and 7j in moderate to excellent yields (65–84%). Interestingly, the electron deficient aldehyde led to synthesis of 7b in 45% yield, but thiophene-2-carbaldehyde furnished the desired product 7i in 75% yield (Figure 2). To illustrate the robust nature of this protocol, reactions of A’ with p-bromobenzaldehdye were conducted on 10 g scale, resulting in smooth transformation into the olefin-pyridone hybrid 7h (67%). X-ray crystallographic analysis of the stereochemistry of 7i revealed that the product is exclusively composed of the Z-enantiomer. This can be attributed to the fact that it is more stable than the E-enantiomer because of the absence of steric repulsion between the aryl and pyridone moieties.

Figure 2.

A variety of electron-rich and electron-deficient aldehydes were used to generate the desired olefins in 45–84% yield.

Antiproliferative activity of compounds

Ten novel compounds were evaluated for their antiproliferative activity against a human melanoma cell line (A375) and against primary human melanoma that metastasized to the brain (35404); human fibroblasts were used to assess compound toxicity towards non-cancerous cells. We chose these melanoma cells, which originate from metastatic tumors, because they are known to overexpress estrogen receptor α (ER-α) and PGK1.^[14,21–23] For each compound, we calculated the concentration that induced 50% growth inhibition (IC50) of each cell type (Table 2). An IC50 value of <10 μM indicates therapeutically relevant antiproliferative activity. We identified 2 compounds 7b (IC50=3.5±3.1 μM) and 7c (IC50=4.3±3.5 μM) – that had therapeutically relevant IC50 values toward A375 human melanoma cells.^[24]

Table 2.

Antiproliferative activity of investigated compounds.

Compound	Antiproliferative activity (IC50, μM)			SI
	A375 cells	35404 cells	Fibroblasts
7a	IN	IN	IN	n/a
7b	3.5±3.1	>10	>10.0	>3
7c	4.3±3.5	>10	>10.0	>2
7d	IN	IN	IN	n/a
7e	IN	IN	IN	n/a
7f	IN	IN	IN	n/a
7g	>10	0.4±0.2	8.1±3.6	>20
7h	IN	IN	IN	n/a
7i	>10	>10	>10	n/a
7j	>10	>10	>10	n/a
Colchicine	9±4 [nM]	-	-	n/a

IC50 values are expressed as mean±standard deviation (n=4 each). IN: inactive; SI: selectivity index.

Clinical trials of novel drug-like agents may fail to a large degree because cell lines might not accurately replicate tumor heterogeneity.^[25] Therefore, we tested the activity of our compounds against patient-derived melanoma that metastasized to the brain (35404 cells). Only compound 7g had therapeutically relevant activity (IC50=0.4±0.2 μM) against 35404 cells.

Cell viability curves for the most potent compounds (i.e., compounds 7b, 7c, and 7g) are shown in Figure 3. Of note, analysis of these 3 cell viability curves revealed that complete cell death was not achieved at the highest clinically relevant concentration that was tested (10 μM), which likely resulted in relatively high standard deviations (SDs).

Figure 3.

Effects of the most potent compounds (i.e., 7b, 7c, and 7g) on viability of melanoma cells in vitro. The graphs represent sensitivity of A375 cells (human melanoma cell line) or 35404 cells (primary melanoma metastasized to brain) to compounds 7b, 7c, and 7g. Cells were treated with 0.1% DMSO (100% viability; negative control) or increasing concentrations of tested compounds. Cell viability was determined with MTT assays; results are presented as mean±SD (n=4) (detailed in Materials and Methods).

For compounds 7b, 7c, and 7g, IC50 values of the 2 types of cancer cells were compared with those of human fibroblasts to calculate the selectivity index (SI), which is an initial indicator of a compound’s therapeutic potential. All 3 compounds had favorable SI values (i.e., >1); most notably, compound 7g had SI >20. This is important because a high SI value indicates that a compound’s efficacy toward cancer cells differs greatly from its toxicity toward normal cells; therefore, treatment with the compound may affect cancer cells to a much greater extent than normal cells.

Compounds 7b and 7c induce DNA fragmentation in A375 cells

To further investigate the mechanism underlying the activity of novel compounds 7b and 7c toward A375 cells, we used flow cytometry to measure DNA content and DNA fragmentation. A375 cells were treated with 0.2% DMSO (control) or with 7b or 7c at concentrations equal to 5 x IC50 values (Table 2) for 24, 48, or 72 h. Cells were briefly stained with propidium iodide and analyzed with flow cytometry to evaluate DNA content; dead cells were quantified as those with sub-G1 DNA (<2 N). The full set of representative cytograms is shown in Figure 4A, and a graphical representation of cells in different phases of the cell cycle (summarized as the mean of 3 biological replicates) is presented in Figure 4B. After 72 h, both compounds 7b and 7c induced statistically significant increases in the amount of Sub-G1 DNA content, relative to the control treatment (Figure 4). Statistically significant DNA fragmentation was observed as early as after 24 h of treatment with 7b, and it increased in a time-dependent manner (Figure 4). Moreover, compound 7b induced transient G2 and/or M phase arrest at 24 h, compared with control treatment; some cells died, and others divided and then went into G1 arrest at 48 h and 72 h. Overall, these findings suggest that hybrid olefin-pyridinone derivatives may predominantly use a mechanism of action similar to that of tamoxifen (i.e., ER-α antagonist) or ilicicolin H (inhibition of PGK1 signaling pathway), which both induce cell cycle arrest at G1.^[26–28]

Figure 4.

Effects of compound 7b or 7c on cell cycle of A375 human melanoma cell line. A. Representative cytograms of A375 cells incubated with DMSO (0.2%; control), 7b (17.5 μM), or 7c (21.5 μM); compounds were used at concentrations equal to 5x respective IC50 values (Table 2). After 24, 48, or 72 h of treatment, cells were subjected to propidium iodide staining and flow cytometry. B. Bar graph representation of the distribution of A375 cells in different phases of the cell cycle, or with sub-G1 DNA. Data are shown as mean±SD (n=3); *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Compounds 7b and 7c attenuate migration of A375 cells

We used a wound-healing assay to examine how treatment with compound 7b or 7c affects the migration of A375 melanoma cells. Primary 35404 metastatic melanoma cells were not used in this study because it was not possible to culture these cells to a confluent monolayer, which is necessary for the assay. The wound (scratch) was created on a confluent monolayer of A375 cells, and cells were allowed to migrate for 54 h in the presence of DMSO (control) or the compound of interest (Figure 5). Molecule 7b significantly reduced the migration of A375 cells as early as 4 h of treatment (76% vs. 93% of wound surface area for control vs. treatment, respectively) (Figure 5B). For both compounds 7b and 7c, statistically significant inhibition of migration of A375 cells was observed after 20, 48, and 54 h of treatment; greater effects were induced by compound 7b. It should be noted that, due to differences in IC50 values (Table 1), compound 7b was used at a lower concentration than compound 7c, but it induced superior effects against cell migration.

Figure 5.

Compounds 7b and 7c reduced migration of A375 cells. A. Representative images of wound healing of monolayers of A375 cells after treatment with DMSO (0.1%; control), compound 7b (3.5 μM), or compound 7c (4.3 μM); compounds were used at concentrations equal to respective IC50 values (Table 2). Wounds were created on a confluent cell monolayer. Images were taken at 0, 2, 4, 20, 48, and 54 h. B. Quantification of wound healing. Data shown as mean±SD (n=3); **p≤0.01, ***p≤0.001, ****p≤0.0001.

Molecular modeling analysis defines molecular interactions with the potential binding sites

Based on our findings from the cell cycle analysis we have observed that the olefin-pyridinone compounds are potentially behaving as either ER-α or PGK1 antagonists as two olefin-pyridinones (7b and 7c) are inducing substantial G1 arrest over 48 and 72 hours. To gain insight into the atomic-level interactions between the olefin-pyridinone compounds and ER-α, the compounds were further assessed by molecular docking at the binding site of the native ligand (4-hydroxytamoxifen, known antagonist, PDB: 2JF9).^[29] The docking analysis (using AutoDock Vina as described in the experimental section) evaluated different poses of ligands for their nonbonding interactions with the binding pocket of ER-α; the docking score results were shown in S.I. Table 1. Results of the docking analysis suggested that, unlike the native ligand (4-hydroxytamoxifen, brown, Figure 6A and 6B), the olefin core of olefin-pyridinones (i.e., compounds 7b; Figure 6A in gold; and 7c; Figure 6B in purple) does not get deep into the hydrophobic tunnel of the ER-α binding site (Figure 6A and 6B). One of the substituted phenyl rings for compounds 7b and 7c enters the deep tunnel, but the rest of the olefin core fits into the shallow hydrophobic cavity outside of tunnel. Thus, unlike the native ligand that forms two hydrogen-binding interactions between phenyl methoxide and Arg394 and Glu353 (Figure 6A and 6B), the olefin-pyridinone compounds primarily interact with the binding site via hydrophobic interactions, resulting in a better docking score for the native ligand (−9.5 kcal/mol) than for compounds 7b (−6.5 kcal/mol) and 7c (−7.3 kcal/mol).

Figure 6.

Molecular docking analysis of compounds 7b (yellow) and 7c (purple), in comparison to a native antagonist (OHT, brown) at the binding site of ER-α (PDB: 2JF9). A. Binding pose of 7b, and OHT (4-hydroxytamoxifen) at the ER- α binding-site surface. B. Binding pose of 7c, and OHT at the ER- α binding-site surface.

The docking analysis against the binding site of PGK1 (PDB: 2XE7)^[13] revealed that the candidates fit well at the binding site and have interactions with the binding site residues that are significantly better than their interactions with the ER-α binding site (Figure 7A and 7B). Compound 7b forms five hydrogenbonding interactions, including three hydrogen bonding interactions between acryloyl ketonic carbonyl oxygen and backbone NH of Gly373 and Gly395 as well as the side chain NH of Arg38 (Figure 7A). The carbonyl oxygen of the amide is engaged in hydrogen-bonding interaction with Thr377. Fluorine substituent on one of the phenyl rings is responsible for hydrogen bonding interaction with the side chain of Thr167, as well as a halogen bonding with the carbonyl oxygen of Gly166. Compared to compound 7b, compound 7c, which has methoxy substituents on the phenyl rings, retains 5 hydrogen bonds (Figure 7B), including a bifurcated H bonding interactions between acryloyl ketonic carbonyl oxygen and Gly373 backbone NH and Arg38 sidechain NH. Amide carbonyl oxygen retains the hydrogen bonding interaction with Thr377. Methoxy substituents on one of the phenyl rings form a bifurcated H bonding interactions with Arg170 side chain NHs. However, 7c loses the halogen bonding interactions with PGK1 binding site (Figure 7C). The docking scores are shown in S.I. Table 1. Overall, the docking analysis suggest that the olefin-pyridinone derivatives have improved binding interactions with the PGK1 binding site and have a potentially unique pose, as compared to the validated PGK1 inhibitor ilicicolin H, which presumably is due to the bulky pyridinone ring.

Figure 7.

Molecular docking analysis of compounds 7b (gold) and 7c (purple), in comparison with a validated antagonist ilicicolin H (brown) at the binding site of PGK1 (PDB: 2XE7). A. Binding pose of 7b (gold), and ilicicolin H (brown) at the PGK1 binding-site. H-bonding interactions are shown using green dotted lines and halogen bonding interactions with cyan dotted lines. B Binding pose of 7c (purple), and ilicicolin H (brown) at the PGK1 binding-site.

To gain an understanding of the protein-ligand stability and protein structural flexibility of the docked complexes, molecular dynamics (MD) simulations were conducted. MD enables realistic ligand-receptor binding interactions to be examined by modeling macromolecular biological systems, which consist of a drug, a receptor, and a solvated environment containing numerous water molecules. The MD simulations were performed on the complexes obtained from the molecular docking analyses against the targets ER-α and PGK1. A total of six complexes (Figure 8 and Figure 9) were considered for the MD simulation. All the complexes were subjected to a 100 ns production run. The ligand-induced conformational changes were then realized by computing the root means square deviation of the protein-ligand complex, the protein backbone, and the ligands separately during the 100 ns MD simulation. The RMSD plots for ER-α showed that compounds 7b and 7c have stable binding with the receptor, and in the 100 ns simulation course, they were limited to the binding site. Regarding the target, PGK1, the complex RMSD plot revealed that compound 7c was bound to the binding site for around 60 ns but then exited the binding pocket, resulting in an enormous RMSD. The RMSD plots for the MD simulations are shown in Figures 9 and 10.

Figure 8.

RMSD vs simulation time plots for 7b, 7c, and 4-hydroxytamoxifen. A. RMSD vs simulation time plot for ER-α protein in complex with 4-hydroxytamoxiden (green), 7b (red), and 7c (blue). B. RMSD vs simulation time plot for movement of ligands, 4-hydroxytamoxifen (green), 7b (red), and 7c (blue), in their binding site.

Figure 9.

RMSD vs simulation time plots for 7b, 7c, and 4-hydroxytamoxifen. A. RMSD vs simulation time plot for PGK1 protein in complex with ilicicolin-H (green), 7b (red), and 7c (blue). B. RMSD vs simulation time plot for movement of ligands, ilicicolin-H (green), 7b (red), and 7c (blue), in their binding site.

Figure 10.

Binding Free Energies and their components. A. Binding free energies and its components for 4-hydroxytamoxifen, 7b, and 7c in complex with ER-α. B. Binding free energies and its components ilicicolin-H, 7b, and 7c in complex with PGK1.

Through Molecular Mechanics Generalized Born Surface Area (MM-GBSA) calculations it was discovered that compounds 7b and 7c exhibited stable binding free energies with ER-α that were similar to those of 4-hydroxytamoxifen, the control. Compared to compound 7b, which has a binding free energy of −19.82 kcal/mol, compound 7c has a superior binding free energy of −31.02 kcal/mol. In comparison to the control, Illicicolin-H, compound 7b, was shown to exhibit a better binding free energy of −24.28 kcal/mol for PGK1. Compound 7c, on the other hand, had a lower binding free energy of −12.17 kcal/mol, which was consistent with the RMSD analysis. It has been demonstrated that the van der Waals interactions account for a significant portion of the total binding free energy in the protein-ligand complexes of both the targets, ERa and PGK1. Interestingly, it was found that for the compound 7b, electrostatic interactions were the major contributor to its highly stable binding free energy against PGK1. Furthermore, the positive interaction energy due to solvation could have had an overall unfavorable energy contribution.

From MM-GBSA calculations, it was observed that the change in binding free energy was negative, and thus, the binding of the compounds to the targets was a thermodynamically favorable process. The decomposition of the binding free energy into various components has been shown in Figure 10. The results from the MM-GBSA calculation are reported in Table 3. Collectively, Molecular modeling analyses suggests that these compounds may act as ER-α and PGK1 inhibitors, thereby introducing cell cycle arrest in G1 phase.

Table 3.

Total binding free energies of the compounds with the targets, ER-α, and PGK1 calculated through MM-GBSA method. The values in parenthesis is the standard error of the mean (SEM).

Compounds	Target: ER-α (kcal/mol)	Target: PGK1 (kcal/mol)
4-hydroxytamoxifen (control)	−37.72 (0.04)	-
Illicicolin-H (control)	-	−17.74 (0.05)
7b	−19.82 (0.03)	−24.28 (0.05)
7c	−31.02 (0.04)	−12.17 (0.16)

Conclusions

We have designed and synthesized a series of olefin-pyridinone derivatives constituted of crucial moieties from tamoxifen and ilicicolin-H. A short and concise synthetic strategy provided access to these challenging scaffolds in 2 steps and produced good yields. The synthesized library of compounds were tested against human melanoma cell line A375 and patient-derived cells of melanoma that had metastasized to brain. Two compounds, 7b and 7c, demonstrated notable antiproliferative activity against A375 melanoma cells, with IC50 values of 3.5 μM and 4.3 μM, respectively. Another compound, 7g, potently inhibited proliferation of patient-derived metastatic melanoma cells, with sub-μM IC50 (IC50=400 nM) and with a moderate selectivity index (SI>10) with respect to normal human fibroblasts. Cell cycle analysis suggested that this set of compounds induced cell cycle arrest predominantly in G1 phase. Compounds 7b and 7c significantly inhibited migration of metastatic melanoma over a period of 54 h; 7b produced greater effects than 7c. Molecular dynamics simulations and binding free energy calculations with MM-GBSA method suggested that compound 7b may have stronger binding with PGK1 (binding free energy= −24.28 kcal/mol) with multiple hydrogen bonding interactions while moderate binding with ER-α (binding free energy= −19.82 kcal/mol). Complex RMSD vs time plot reveals that compound 7b maintains close proximity with both PGK1 and ER-α binding site unlike compound 7c which exits the PGK1 binding site during the course of the simulation. MM-GBSA binding free energy calculations suggest that compound 7c may have stronger binding for ER-α with binding free energy of −31.02 kcal/mol. Taken together, our results indicate that compounds 7b and 7c are promising lead structures and that further research is warranted to improve the activity of these 2 compounds as new anti-melanoma drug candidates. Further studies of structure-activity relationship and biological evaluations will be reported in due course.

Experimental Section

Chemistry Experimental Methods

General

All reactions were carried out in flame-dried sealed tubes with magnetic stirring. Unless otherwise noted, all experiments were performed under argon atmosphere. All reagents were purchased from Sigma Aldrich, Acros, or Alfa Aesar. Solvents were treated with 4 Å molecular sieves or sodium and were distilled before use. Reaction products were purified with column chromatography (Chem Lab silica gel, 230–400 mesh). ¹H-NMR and ¹³C NMR (Bruker AVHDN at 400 MHz and 100 MHz, respectively) spectra were recorded with tetramethylsilane as an internal standard at ambient temperature, unless otherwise indicated. Chemical shifts are reported in parts per million, and coupling constants are reported as Hz. Splitting patterns were designated as singlet (s), broad singlet (bs), doublet (d), or triplet (t). Splitting patterns that could not be interpreted or easily visualized were designated as multiple (m). Mass spectrometry analyses were done with the 6540 UHD Accurate-Mass Q-TOF LC–MS system (Agilent Technologies) equipped with Agilent 1290 LC system (obtained by Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Uttar Pradesh 203207, India).

General protocol for synthesis of substituted (Z)-4-aryl-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one

To a stirred solution of 6-methylfuro[3,4-c]pyridine-3,4(1H,5H)-dione (200 mg; 1.21 mmol) in 10 mL ethanol, piperidine (516 mg; 6.06 mmol) was added, followed by the corresponding aldehyde (2.42 mmol), under N₂ atmosphere at room temperature. The reaction mixture was stirred at 90° C for 16 h, allowed to cool to room temperature, and evaporated under vacuum. The resulting residue was purified with chromatography on silica gel to afford substituted (Z)-4-aryl-6-methyl-3-(piperidine-1-carbonyl)pyridine-2(1H)-one.

(Z)-4-(2,3-di-o-tolylacryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7a)

With the general protocol, 2-methylbenzaldehyde (290 mg, 2.42 mmol) afforded 381 mg (yield 84%) of compound 12b as a pale brown solid. ¹H-NMR (400 MHz; CDCl3): δ 7.77 (s, 1H), 7.20–7.11 (m, 5H), 6.81 (d, J=8 Hz, 2H), 6.70 (d, J=8 Hz, 1H), 6.16 (s, 1H), 3.63 (bs, 2H), 3.32 (bs, 2H), 2.37 (s, 3H), 2.20 (s, 6H), 1.61 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 195.80, 163.90, 162.16, 146.59, 134.28, 133.45, 130.30, 129.59, 129.07, 128.22, 127.78, 126.06, 125.51, 123.59, 113.97, 104.65, 48.08, 42.61, 31.03, 26.05, 25.45, 24.57, 20.09, 19.14. HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for (C₂₉H₃₀N₂O₃) 455.2329, found 455.2385.

(Z)-4-(2,3-bis(3-fluorophenyl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7b)

With the general protocol, 3-fluorobenzaldehyde (300 mg, 2.42 mmol) afforded 207 mg (yield 45%) of compound 12f as a dark brown powder. ¹H-NMR (400 MHz; CDCl3): δ 7.33 (s, 1H), 7.32–7.26 (m, 1H), 7.09 (d, J=8 Hz, 1H), 7.01–6.88 (m, 4H), 6.81 (d, J= 8 Hz, 1H), 6.66 (d, J=8 Hz, 1H), 6.10 (s, 1H), 3.52 (bs, 2H), 3.19 (bs, 2H), 2.33 (s, 6H), 1.48 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 195.81, 162.82, 160.77, 150.52, 146.18, 143.43, 138.28, 135.47, 134.96, 134.88, 129.46, 128.93, 128.85, 125.74, 124.66, 116.21, 116.04, 115.99, 115.82, 114.62, 103.91, 47.13, 41.78, 28.68, 25.04, 24.41, 23.42, 21.31, 18.21. HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for (C₂₇H₂₄F₂N₂O₃) 463.1828, found 463.1829

(Z)-4-(2,3-bis(4-methoxy-3-methylphenyl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7c)

Following the general protocol 4-methoxy-3-methylbenzaldehyde (363 mg, 2.42 mmol) afforded compound 12g in 392 mg (yield 71%) brown solid. ¹H-NMR (400 MHz; CDCl3): δ 7.32 (s, 1H), 7.11 (s, 1H), 7.04 (d, J=8 Hz, 1H), 7.03 (s, 1H), 7.00 (s, 1H), 6.94 (s, 1H), 6.91 (d, J=8 Hz, 1H), 6.85 (d, J=8 Hz, 1H), 6.59 (d, J=8 Hz, 1H), 6.15 (s, 1H), 3.86 (s, 3H), 3.78 (s, 3H), 3.61 (bs, 2H), 3.27 (bs, 2H), 2.36 (s, 3H), 2.20 (s, 3H), 2.04 (s, 3H), 1.55 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 196.26, 164.40, 159.26, 157.58, 152.12, 146.92, 146.78, 136.98, 134.24, 131.98, 130.47, 128.35, 127.07, 126.56, 122.74, 110.37, 109.57, 105.20, 63.77, 55.32, 48.13, 42.66, 26.00, 25.40, 24.47, 19.18, 16.25, 16.04. HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for (C₃₁H₃₄N₂O₅K) 553.2149, found 553.2169.

(Z)-4-(2,3-bis(3,5-dimethylphenyl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7d)

With the general protocol, 3,5-dimethylbenzaldehyde (329 mg, 2.42 mmol) afforded 294 mg (yield 61%) of compound 12i as half white solid. ¹H-NMR (400 MHz; CDCl3): δ 7.32 (s, 1H), 6.99 (s, 1H), 6.86 (s, 1H), 6.83 (s, 2H), 6.69 (s, 2H), 6.14 (s, 1H), 3.61 (bs, 2H), 3.28 (bs, 2H), 2.28 (s, 6H), 2.16 (s, 3H), 2.11 (s, 6H), 1.60 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 195.33, 164.21, 162.03, 151.82, 147.02, 146.93, 139.41, 138.18, 137.55, 134.91, 134.07, 131.67, 129.78, 129.15, 127.29, 123.00, 105.14, 63.83, 48.14, 42.69, 26.01, 25.39, 24.50, 21.31, 21.09, 19.19. HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for (C₃₁H₃₄N₂O₃) 483.2682, found 483.2688.

(Z)-4-(2,3-bis(2,4,5-trimethoxyphenyl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7e)

With the general protocol, 2,4,5-trimethoxybenzaldehyde (474 mg, 2.42 mmol) afforded 454 mg (yield 75%) of compound 12j as a yellow solid. ¹H-NMR (400 MHz; DMSO-d6): δ 7.82 (s, 1H), 6.90 (s, 1H), 6.57 (s, 1H), 6.52 (s, 1H), 6.42 (s, 1H), 6.22 (s, 1H), 3.89 (s, 6H), 3.82 (s, 3H), 3.78 (s, 3H), 3.77 (bs, 2H), 3.66 (s, 3H), 3.32 (bs, 2H), 3.31 (s, 3H), 2.18 (s, 3H), 1.62 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 196.10, 164.29, 162.23, 154.14, 152.83, 151.65, 151.34, 149.68, 146.77, 146.03, 143.91, 142.35, 123.61, 116.83, 115.49, 114.89, 112.08, 110.36, 105.45, 98.22, 96.45, 96.36, 56.93, 56.72, 56.64, 56.58, 56.36, 56.22, 55.89, 55.49, 48.06, 42.36, 26.03, 25.47, 24.62, 19.11. HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for (C₃₃H₃₈N₂O₉) 607.2653, found 607.2663.

(Z)-4-(2,3-bis(4-chlorophenyl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7f)

With the general protocol, 4-chlorobenzaldehyde (338 mg, 2.42 mmol) afforded 370 mg (yield 75%) of compound 12k as a light brown powder. ¹H-NMR (400 MHz; CDCl3): δ 7.41 (s, 1H), 7.38 (d, J=8 Hz, 2H), 7.22 (d, J=8 Hz, 2H), 7.18 (d, J=8 Hz, 2H), 7.03 (d, J=8 Hz, 2H), 6.15 (s, 1H), 3.61 (bs, 2H), 3.26 (bs, 2H), 2.40 (s, 3H), 1.27 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 195.45, 164.01, 161.88, 151.58, 147.39, 144.39, 138.77, 135.98, 132.40, 131.99, 131.41, 129.21, 128.74, 123.29, 104.84, 48.14, 42.76, 26.05, 25.45, 24.42, 19.21. HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for (C₂₇H₂₄Cl₂N₂O₃) 495.1237, found 495.1293

(Z)-4-(2,3-bis(3-chloro-4-methylphenyl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7g)

With the general protocol, 3-chloro-4-methylbenzaldehyde (372 mg, 2.42 mmol) afforded 438 mg (yield 84%) of compound 12 l as a brown solid. ¹H-NMR (400 MHz; CDCl3): δ 7.33 (s, 1H), 7.26 (s, 2H), 7.24–7.23 (m, 1H), 7.14 (s, 1H), 7.05 (d, J=8 Hz, 1H), 7.04 (d, J=8 Hz, 1H), 6.82 (d, J=8 Hz, 1H), 6.13 (s, 1H), 3.60 (bs, 2H), 3.26 (bs, 2H), 2.4 (s, 6H), 2.31 (s, 3H), 1.26 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 195.54, 163.95, 161.98, 151.52, 147.21, 144.74, 138.40, 138.26, 136.24, 134.76, 134.44, 133.64, 133.15, 131.48, 131.42, 130.82, 130.17, 128.66, 128.20, 123.26, 104.88, 48.12, 42.75, 29.69, 26.06, 25.44, 24.46, 20.03, 19.97, 19.22. HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for (C₂₉H₂₈Cl₂N₂O₃) 523.1570, found 523.1579.

(Z)-4-(2,3-bis(4-bromophenyl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7h)

With the general protocol, 4-bromobenzaldehyde (445 mg, 2.42 mmol) afforded 461 mg (yield 79%) of compound 12m as a brown solid. ¹H-NMR (400 MHz; CDCl3): δ 7.36 (s, 1H), 7.19 (d, J= 8 Hz, 2H), 7.06 (d, J=8 Hz, 2H), 6.93 (d, J=8 Hz, 2H), 6.69 (d, J= 8 Hz, 2H), 6.12, (s, 1H), 3.84 (s, 2H), 3.76 (s, 2H), 2.37 (s, 3H), 1.55 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 196.15, 164.20, 162.04, 160.86, 159.37, 152.22, 146.88, 145.63, 137.06, 132.91, 131.23, 127.46, 127.03, 123.10, 114.40, 113.81, 105.09, 55.26, 55.22, 48.09, 42.64, 26.04, 25.46, 24.51, 19.17. HRMS (ESI-TOF) m/z: [M+4H]⁺ calculated for (C₂₇H₂₄Br₂N₂O₃) 585.0280, found 585.0282.

(Z)-4-(2,3-bis(4-bromothiophen-2-yl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7i)

With the general protocol, 4-bromothiophene-2-carbaldehyde (459 mg, 2.42 mmol) afforded 445 mg (yield 75%) of compound 12n as a brown solid. ¹H-NMR (400 MHz; CDCl3): δ 13.06 (bs, 1H), 7.72 (s, 1H), 7.45 (s, 1H),7.32 (s, 1H), 7.27 (s, 1H), 7.00 (s, 1H), 6.09 (s, 1H), 3.60 (bs, 2H), 3.30 (bs, 2H), 2.39 (s, 3H), 1.60 (m, 6H). ¹³C NMR (125 MHz; DMSO-d6): δ 193.73, 163.76, 159.47, 138.46, 138.03, 135.34, 131.45, 131.32, 129.34, 127.23, 109.48, 108.86, 43.67, 25.24, 24.95, 23.50, 20.18, 20.54, 19.21. HRMS (ESI-TOF) m/z: [M+4H]⁺ calculated for (C₂₃H₁₉Br₂N₂O₃S₂) 596.9355, found 596.9359.

(Z)-4-(2,3-bis(4-propoxyphenyl)acryloyl)-6-methyl-3-(piperidine-1-carbonyl)pyridin-2(1H)-one (7j)

With the general protocol, 4-propoxybenzaldehyde (397 mg, 2.42 mmol) afforded 439 mg (yield 81%) of compound 7j as a dark yellow solid. ¹H-NMR (400 MHz; CDCl3): δ 7.35 (s, 1H), 7.18 (d, J= 8 Hz, 2H), 7.05 (d, J=8 Hz, 2H), 6.91 (d, J=8 Hz, 2H), 6.68 (d, J= 8 Hz, 2H), 6.10 (s, 1H), 3.96–3.93 (t, J=4 Hz, 2H), 3.89–3.85 (t, J= 4 Hz, 2H), 3.61 (bs, 2H), 3.28 (bs, 2H), 2.35 (s, 3H), 1.86–1.76 (m, 4H), 1.54 (m, 6H), 1.07–0.98 (m, 6H). ¹³C NMR (100 MHz; CDCl3): δ 196.17, 164.18, 162.11, 160.48, 158.95, 152.30, 146.78, 145.70, 136.97, 132.94, 131.21, 127.29, 126.85, 123.11, 114.93, 114.28, 69.51, 69.46, 48.09, 42.62, 26.05, 25.46, 24.53, 22.64, 22.45, 19.16, 10.57, 10.43. HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for (C₃₃H₃₈N₂O₅) 543.2853, found 543.2885.

Biological Experiments

Cells and culturing conditions

Human malignant melanoma cell line A375 was cultured routinely in Dulbecco’s Modified Eagle Medium (DMEM) (cat. no. 10–013-CV; Corning, Manassas, VA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (cat. no. FP-0500-A; Atlas Biologicals, Fort Collins, CO, USA), 5% L-glutamine (cat. no. 25–005-Cl; Corning), 5% non-essential amino acids (cat. no. 11140–050; Gibco), and 1 mM β-mercaptoethanol. Human primary dermal fibroblasts were purchased (cat. no. PCS-201–012; American Type Culture Collection [ATCC]) and maintained in fibroblast basal medium (cat. no. CS-201–030; ATCC) supplemented with Fibroblast Growth Kit-Low serum (cat. no. PCS-201–041, ATCC) according to manufacturer recommendations. All cells used in this study were maintained in a humidified incubator at 37°C and 5% CO₂. Mycoplasma testing and cell validation of A375 cells were performed with short tandem repeat profiling in February 2022 by Genetica Laboratories (Burlington, NC, USA). The cell line was confirmed as authentic by 100% match with the known reference profile^[30] and was verified to be negative for mycoplasma.

Generation and culturing of primary metastatic melanoma to brain cells

Primary patient-derived metastatic tissue sample (CI000003404) was obtained from the Tissue Biorepository and Procurement Core (University of Arkansas for Medical Sciences [UAMS], Little Rock, AR, USA) within a few hours of surgical removal; consent was obtained from all patients before surgery (IRB# 228443). Upon receipt, the tumor was digested, cultured, and samples were biobanked. Research biobanks collect samples and data of human origin for their own research or that of third parties. For digestion, tissue was placed in 5 ml aliquot of digestion buffer, consisting of 100 mg collagenase Type II (cat. no. LS004176; Worthington-Biochem Corp., Lakewood, NJ, USA), 100 mg collagenase Type IV (cat. no. 17104–019; Gibco), 200 mg DNAse (cat. no. DN25–1G; Sigma),100 ml DMEM, 5 ml Pen-Strep (cat. no. 14140–122; Gibco),^[31] the digestion buffer was stored in 5 ml aliquots at −20°C until used. The sample was incubated overnight at 4°C on an orbital rotator. The solution was strained through a 70-μm filter and washed with 3 ml of Dulbecco’s phosphate buffer saline (DPBS; cat. no. 21–030-CM; Corning), followed by a Ficoll-Paque step (35 min, 300g, room temperature [RT]). The interface was washed, and red blood cells were lysed (10 min, RT; followed by 5 min, 500g). The pellet was washed with 6 ml DPBS (10 min, 100 g) and resuspended in 10 ml DPBS to count cell number (Celldrop XL; DeNovix Inc, Wilmington,DE, USA). Dissociated tumor cells were then cultured. They were seeded in tissue culture plates/flasks in a mixture of RPMI1640 (cat. no. 21870–076; Gibco) supplemented with 10% FBS (cat. no. 35–011-CV; Corning), 2.5 g/L glucose (cat. no. A24940–01; Gibco), 4 mmol/L L-glutamine (cat. no. 25030–081; Gibco), 100 U/mL penicillin and 100 U/mL streptomycin (cat. no. 15140–122; Gibco). Cells were maintained in a humidified incubator at 37°C and 5% CO₂. Cells were passaged and expanded after reaching 80% to 90% confluency. Cells were considered a cell line upon reaching passage 9.

Cell viability assay

To evaluate cell viability, we used an assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).^[32] Investigated compounds were dissolved in cell-culture grade DMSO (cat. no. BP231–1, Fisher). All cells used in this study were seeded at a density of 2×10³ cells/well in 96-well plates (TPP, Trasadingen, Switzerland) in 100 μl of corresponding complete medium. After 24 h of incubation, cells were treated for 72 h with compounds at concentrations up to 10 μM or with 0.1% DMSO (negative control). After treatment, MTT solution (10 μl of 5 mg/ml) was added to each well, and the plates were incubated for an additional 4 h in a humidified incubator at 37°C and 5% CO₂. Medium then was removed, and 150 μl of DMSO was added to each well; absorbance at 540 nm was measured with a BioTek plate reader. As an index of cell viability, inhibition of formation of colored MTT formazan was determined. IC50 values were determined with non-linear regression analysis (GraphPad Prism 9 for Windows).

Flow cytometry analysis of cell cycle

A375 cells (10⁵ cells/well for treatment with DMSO; 2×10⁵ cells/well for treatment with a compound) were seeded in 6-well plates (TPP) and allowed to adhere for 24 h in a humidified incubator at 37°C and 5% CO₂. After 24 h incubation, the medium was replaced, and cells were treated with 0.2% DMSO, 17.5 μM of compound 7b, or 21.5 μM of compound 7c (concentrations are equal to 5×IC50 values, as shown in Table 2). After 24, 48, or 72 h, cells were harvested, washed with DPBS, fixed with 2 ml of 70% ice-cold ethanol, and stored at 4°C prior to flow cytometric analysis. Cells were centrifuged, stained with 300 μl propidium iodide/RNase staining buffer (BD Biosciences, San Jose, CA, USA), and stored in the dark at RT for 1 h. DNA content was quantitated with FacsAria IIIu Flow Cytometer (BD Biosciences), and data were analyzed with FlowJo software. Three biological replicates were performed for each treatment.

Wound-healing assay

A375 cells were seeded in 60-mm petri dishes (3×10⁵ cells/dish) and allowed to adhere for 120 h in a humidified incubator at 37°C and 5% CO₂, resulting in a confluent cell monolayer. After removing the complete growth medium, cells were washed with DPBS and incubated overnight in fresh medium without FBS. The medium was removed, and cross-shaped scratches (wound areas) in the cell monolayer were made with a sterile 1250-μl pipette tip. Next, cells were washed twice with DPBS and incubated in medium containing 0.1% DMSO, 3.5 μM of compound 7b, or 4.3 μM of compound 7c (concentrations are equal to respective IC50 values, presented in Table 2). The scratch area was photographed with inverted phase contrast microscopy (Nikon Eclipse Ts2) directly at baseline (0 h) and after 2, 4, 20, 48, and 54 h. Three biological replicates were performed for each treatment, and 7 measurements were taken for each condition and time point; mean±SD was calculated. The wound area at each time point was divided by the area at 0 h to obtain the measure of wound closure. The wound area was defined as 100% at 0 h for each treatment condition.

Statistical Analysis

GraphPad Prism 9 for Windows was used for statistical analyses. Unpaired t-test with Welch’s correction was used to determine significance, and p values <0.05 were considered significant. Data are presented as a mean±SD.

Molecular Docking

The three-dimensional structures of compounds 7a–7j were created via MM2 energy minimization using Chem3D modeling software. The three-dimensional structures of the targets, estrogen receptor alpha (ER-α) [PDB ID: 2JF9] and phosphoglycerate kinase 1 (PGK1) [PDB ID: 2XE7] were downloaded from the RCSB Protein Databank. By employing the GROMOS96 force field to minimize the energy of the protein structures, bad contacts, and unfavorable torsion angles were removed using the Swiss PDB-Viewer 4.1 program^[33] The AutoDock Vina software, version 1.1.2, was used for all docking runs. MGL Tools were used to generate the structure format (PDBQT) for AutoDock Vina.^[34] The binding site of the native ligand (4-hydroxytamoxifen) found in PDB structure 2JF9 served as the centre of the grid box for ER-α. The grid box for PGK1 was positioned at the Iliicicolin-H binding site, as reported by Jing Li. et al.^[13] The box dimensions in both instances were 25×25×25 Å. The docking process consisted of eight independent runs per ligand, and twenty binding modes were generated. The results were represented with the best binding affinity. The Biovia Discovery Studio visualizer was used to illustrate the interactions that were occurring between the ligands and the targets.

Validation of docking procedure

The docking procedure was validated by a control study to confirm that the docked pose of the native ligand is in alignment with the crystal structure pose. The bound ligand, OHT, in crystal structures 2JF9 was redocked to the preprocessed and prepared protein, keeping the same grid box. The score for this docking was used as a standard value against which the scores for the drug like agents were compared. For PGK1 crystal structure (2XE7), molecular docking of ilicicolin H was performed using the docking parameters and crucial amino acid residues as described by Jing Li et al to make sure that our findings are in concert with the reported pose.^[13] The control docking was performed with AutoDock Vina as described above.

Molecular Dynamics Simulation

The initial coordinates of the protein-ligand complexes were obtained from the molecular docking. The MD simulations were performed using the Groningen Machine for Chemical Simulations (GROMACS) software with the CHARMM36 force field.^[35–36] The CHARMM general force field (CGenFF) was employed for the ligand molecules.^[37] The structure was placed in a dodecahedron simulation box with a periodic boundary condition filled with TIP3P model water molecules. Next, Na⁺ and Cl⁻ ions were added to the system to neutralize it. Following appropriate parametrization, the system’s geometries were minimized through 5,000 steps by the steepest descent and 10,000 steps by the conjugate gradient method in order to remove any undesirable interactions and relax the system. Under the NVT ensemble, the system was gradually annealed for 50 ps in order to reach an optimal temperature of 300 K. Following that, one ns density equilibration was carried out at NPT ensemble conditions (constant temperature 300 K and constant pressure 1.0 atm) in order to get the uniform density. A Langevin thermostat with a collision frequency of 2 ps and a Berendsen barostat with a pressure relaxation time of 1 ps were employed, respectively, to maintain the constant temperature and pressure. The van der Waals interactions in the MD simulation used the particle mesh Ewald technique to describe long-range electrostatic interactions with a cut-off of 1.2 nm. Next, the complexes were investigated using production dynamics spanning 100 nanoseconds.

Binding Free Energy calculations

The binding free energy calculations were performed on the last 5000 frames (50 ns) obtained from the MD simulation using the MM-GBSA (Molecular Mechanics Generalized Born Surface Area) method through the gmx_MMPBSA tool.^[38] These calculations include the energetic terms for van der Waals contributions from molecular mechanics, the electrostatic energy and electrostatic contribution to the solvation-free energy calculated by the Generalized Born equation, and the nonpolar contribution to the solvation-free energy calculated by an empirical model. The water molecules and the added ions present in the system had been removed before the MM-GBSA analysis. Binding free energies (DG_Total) of the ligands with the target proteins were calculated as follows:

$$\begin{gathered} {\text{DG}}_{\text{Total}}={\text{DG}}_{\text{gas}}+{\text{DG}}_{\text{solv}} \\ \text{where},{\text{DG}}_{\text{gas}}{\text{=DE}}_{\text{vdw}}{\text{+DE}}_{\text{ele}} \\ \text{and}{\text{DG}}_{\text{solv}}{\text{=DE}}_{\text{polar}}{\text{+DE}}_{\text{non}-\text{polar}} \end{gathered}$$

DG_Total is the sum of gas phase molecular mechanics energy (DG_gas) and solvation-free energy, DG_solv. Both DG_gas and DG_solv are further divided into van der Waals (DE_vdw) and electrostatic energy (DE_ele) in the gas phase and polar (DE_polar) and nonpolar (DE_non-polar) contributions to the solvation-free energy. The detailed protocol of this method has already been discussed elsewhere.^[39]

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.